*Target (protein/gene name): Dihydrofolate Reductase *NCBI Gene # or RefSeq#:14966432 *Protein ID (NP or XP #) or Wolbachia#: YP_007611485.1 *Organism (including strain):M. tuberculosis Etiologic Risk Group (see link below): RG3 *Background/Disease Information (sort of like the Intro to your Mini Research Write up):Mycobacterium tuberculosis is a pathogenic bacteria that is the causative agent of tuberculosis. The bacteria has an unusual, waxy coating that prevents it from being effectively tested with Gram staining and instead acid-fast detection is used. The pathogen is highly aerobic and primarily infects the lungs of mammals. Tuberculosis is an infectious disease that in many cases is legal. It is spread through the air by coughing, sneezing, or transmission of respiratory fluids from an infected person. Symptoms include chronic cough, blood-tinged sputum, fever, night sweats, and weight loss. Diagnosis of TB is dependent on radiology, usually a chest X-ray. Tuberculosis is much more active in the developing world with about 80% of the population in many Asian and African countries testing positive for tuberculosis, while only about 5-10% test positive in the U.S. population. The emergence of multiple drug-resistant tuberculosis (MDR-TB) is causing a big push for the development of new drugs for this infectious disease. Essentiality of this protein: Essential Complex of proteins?: No Druggable Target: Yes *EC#: 1.5.1.3 Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3&Suchword=&organism[]=Mycobacterium+tuberculosis&show_tm=0
Fig 1. Enzyme schematic of dihydrofolate reductase in M. tuberculosis.
Expression Information (has it been expressed in bacterial cells): Yes in E.coli cells Purification Method: metal-chelate affinity chromatography [1] Image of protein (PyMol with features delineated and shown separately):
Fig 2. M. tuberculosis DHFR (PDB ID: 1DG8) shown with NADPH in active site. Carbon of NADPH shown in pink, hydrophillic residues of protein shown with carbon in green, hydrophobic residues of protein shown with carbon in yellow, water shown as red spheres, oxygen in red, nitrogen in blue, and phosphorus in orange.
*Amino Acid Sequence:
MVGLIWAQATSGVIGRGGDIPWRLPEDQAHFREITMGHTIVMGRRTWDSLPAKVRPLPGRRNVVLSRQADFMASGAEVVG SLEEALTSPETWVIGGGQVYALALPYATRCEVTEVDIGLPREAGDALAPVLDETWRGETGEWRFSRSGLRYRLYSYHRS *length of your protein in Amino Acids: 159 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17640.0 Molar Extinction coefficient of your protein at 280 nm wavelength: 40450 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html)
Graph Target Discovery.gif
*CDS Gene Sequence (paste as text only): GTGACGATGGTGGGGCTGATCTGGGCTCAAGCGACATCGGGTGTCATCGGCCGCGGCGGCGACATCCCCT GGCGCTTGCCCGAGGACCAGGCGCATTTCCGGGAGATCACCATGGGGCACACGATCGTGATGGGCCGGCG CACATGGGATTCGCTGCCGGCTAAAGTCCGGCCGCTGCCCGGCCGGCGAAATGTCGTACTGAGCCGCCAA GCTGACTTTATGGCCAGCGGGGCTGAGGTTGTCGGTTCACTCGAGGAGGCGCTGACCAGCCCGGAGACGT GGGTGATCGGAGGCGGACAAGTCTATGCGCTGGCGCTGCCGTACGCGACCAGATGTGAGGTTACCGAGGT CGACATCGGCCTGCCGCGCGAAGCCGGTGACGCGCTGGCCCCCGTGCTGGACGAGACATGGCGGGGCGAG ACGGGGGAGTGGCGCTTCAGCCGGTCCGGGTTGCGGTACCGGTTGTACAGCTACCACCGCTCATGA *GC% Content for gene: 67.08% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
[1] Argyrou, Argyrides; Vetting, Matthew W.; Aladegbami, Bola; Blanchard, John S., Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nature Stuctural & Molecular Biology,2006. 13. 408-13.
*NCBI Gene # or RefSeq#: 14966432
*Protein ID (NP or XP #) or Wolbachia#: YP_007611485.1
*Organism (including strain): M. tuberculosis
Etiologic Risk Group (see link below): RG3
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium tuberculosis is a pathogenic bacteria that is the causative agent of tuberculosis. The bacteria has an unusual, waxy coating that prevents it from being effectively tested with Gram staining and instead acid-fast detection is used. The pathogen is highly aerobic and primarily infects the lungs of mammals. Tuberculosis is an infectious disease that in many cases is legal. It is spread through the air by coughing, sneezing, or transmission of respiratory fluids from an infected person. Symptoms include chronic cough, blood-tinged sputum, fever, night sweats, and weight loss. Diagnosis of TB is dependent on radiology, usually a chest X-ray. Tuberculosis is much more active in the developing world with about 80% of the population in many Asian and African countries testing positive for tuberculosis, while only about 5-10% test positive in the U.S. population. The emergence of multiple drug-resistant tuberculosis (MDR-TB) is causing a big push for the development of new drugs for this infectious disease.
Essentiality of this protein: Essential
Complex of proteins?: No
Druggable Target: Yes
*EC#: 1.5.1.3
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3&Suchword=&organism[]=Mycobacterium+tuberculosis&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
--http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number.html
--http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/dihydrofolatereductase.pdf
--Reagents: (Sigma)
potassium phosphate buffer : 100g for $19.90
ß-NADPH: ........................... 15VL for $345.00
DHFA: ..................................10 mg for $71.60
BSA:........................................ 5g for $159.00
DHFR Enzyme solution:.. .25 units for $241.50
Structure Available (PDB or Homology model)
-- 1DG8
Current Inhibitors:
--3-([(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)methyl]amino)-4-(2,5-dimethoxyethoxy)phenol
--4,5-dideoxy-5-(2,6-diamino-5-phenylpyrimidin-4-yl)-D-erythro-pentitol
--6-([(2,5-dipropoxyphenyl)amino]methyl)-5-methylpyrido[2,3-d]pyrimidine-2,4-diamine
--6-([(2-butoxy-5-ethoxyoxyphenyl)amino]methyl)-5-methylpyrido[2,3-d]pyrimidine-2,4-diamine
--6-([(2-butoxy-5-propoxyphenyl)amino]methyl)-5-methylpyrido[2,3-d]pyrimidine-2,4-diamine
--6-([(2-ethoxy-5-butoxyphenyl)amino]methyl)-5-methylpyrido[2,3-d]pyrimidine-2,4-diamine
--Isoniazid
--methotrexate
--Pyrimethamine
--trimethoprim
--tripeptide inhibitor WYY
Expression Information (has it been expressed in bacterial cells): Yes in E.coli cells
Purification Method: metal-chelate affinity chromatography [1]
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
MVGLIWAQATSGVIGRGGDIPWRLPEDQAHFREITMGHTIVMGRRTWDSLPAKVRPLPGRRNVVLSRQADFMASGAEVVG SLEEALTSPETWVIGGGQVYALALPYATRCEVTEVDIGLPREAGDALAPVLDETWRGETGEWRFSRSGLRYRLYSYHRS
*length of your protein in Amino Acids: 159
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17640.0
Molar Extinction coefficient of your protein at 280 nm wavelength: 40450
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html)
*CDS Gene Sequence (paste as text only):
GTGACGATGGTGGGGCTGATCTGGGCTCAAGCGACATCGGGTGTCATCGGCCGCGGCGGCGACATCCCCT GGCGCTTGCCCGAGGACCAGGCGCATTTCCGGGAGATCACCATGGGGCACACGATCGTGATGGGCCGGCG CACATGGGATTCGCTGCCGGCTAAAGTCCGGCCGCTGCCCGGCCGGCGAAATGTCGTACTGAGCCGCCAA GCTGACTTTATGGCCAGCGGGGCTGAGGTTGTCGGTTCACTCGAGGAGGCGCTGACCAGCCCGGAGACGT GGGTGATCGGAGGCGGACAAGTCTATGCGCTGGCGCTGCCGTACGCGACCAGATGTGAGGTTACCGAGGT CGACATCGGCCTGCCGCGCGAAGCCGGTGACGCGCTGGCCCCCGTGCTGGACGAGACATGGCGGGGCGAG ACGGGGGAGTGGCGCTTCAGCCGGTCCGGGTTGCGGTACCGGTTGTACAGCTACCACCGCTCATGA
*GC% Content for gene: 67.08%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
[1] Argyrou, Argyrides; Vetting, Matthew W.; Aladegbami, Bola; Blanchard, John S., Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nature Stuctural & Molecular Biology,2006. 13. 408-13.See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf